Cystic fibrosis (CF) is a lethal genetic disease caused by the lack of functional cystic fibrosis transmembrane conductance regulator protein (CFTR). There is no cure for CF, and the primary goal in the field is to develop therapeutics to bypass or correct the basic defects in CFTR. CFTR contains two transmembrane spanning domains (TMDs), two nucleotide binding domains (NBDs), and one regulatory region (R) that are translated in the order: TMD1-NBD1-R-TMD2-NBD2. For CFTR, domains each fold cotranslationally and then hierarchically associate to form the functional protein. The most common CF mutation, ?F508, and other CF mutations disrupt multiple steps in this folding pathway. Details about cotranslational CFTR (mis)folding and interactions with cellular protein quality control machinery are limited. CF-causing mutations in TMD1 and NBD1 destabilize the protein prior to completion of CFTR translation. It has been proposed that TMD1 and NBD1 interact during translation, and that this interaction is disrupted by either mutations in TMD1 or the ?F508 mutation in NBD1. Yet, these interactions have not been directly demonstrated experimentally. When quality control proteins initially recognize mutant CFTR as misfolded is also not known. Mechanistic studies of native and mutant CFTR folding during translation and identification of the proteins involved are needed for the targeted development of CF therapeutics. The aims of this proposal are to: 1. Test the hypothesis that interdomain interactions between TMD1 and NBD1 are formed during CFTR translation and that CF mutations disrupt these interactions. A powerful technique designed to site-specifically incorporate a photoreactive probe into a nascent chain of defined length will be used to examine this interdomain interaction during translation of normal and mutant CFTR. Transmembrane span integration and topology will be related to formation of this interaction. 2. Identify a 70kDa protein that interacts specifically with the mutant CFTR during translation. Using the photoreactive probe approach, an unidentified 70kDa protein has been found that interacts preferentially with mutant CFTR early during translation. This protein will be identified using a biochemical fractionation and reconstitution approach. The proposed studies utilize extant methodology in the laboratory to address the basic mechanism of CFTR (mis)folding and its relation to CF, and to examine the poorly understood process of multidomain membrane protein folding.